CAJ | 학술논문

目的建立稳定干涉癌基因Gankyrin的4T1-luc细胞株,探讨Gankyrin对小鼠乳腺癌转移的影响.方法采用慢病毒感染和抗性筛选方法获得稳定干涉Gankyrin的乳腺癌细胞株4T1-luc/shGankyrin,通过蛋白质印迹和实时定量PCR方法分别在蛋白质和mRNA水平检测Gankyrin的干涉效果；选用BALB/c小鼠进行4T1细胞原位种植,利用活体成像技术实时观测小鼠体内肿瘤生长和肿瘤转移情况,并对小鼠肺转移瘤进行病理学分析.结果采用免疫印迹和实时定量PCR,检测稳定敲低Gankyrin 4T1细胞在蛋白质水平和mRNA水平的表达,均明显低于对照细胞,与对照细胞相比,不同干涉序列的4T1细胞mRNA水平分别为对照细胞的4.9％、25.1％、69.8％.利用活体成像进行细胞数与细胞发光强度的检测,选择细胞数与发光强度基本一致的#2细胞株进行小鼠原位种植,其产生的转移瘤进行实时观测,结果显示敲低Gankyrin的4T1细胞诱导的肺转移瘤荧光强度为3.02×106,而对照细胞为10.9×106,同时病理学证实了对照细胞产生的肺转移瘤免疫组织化学Gankyrin染色阳性,而敲低Gankyrin的4T1细胞的肺转移瘤免疫组织化学Gankyrin染色阴性.结论采用慢病毒感染的方法可以有效建立稳定敲低Gankyrin表达的细胞株；Gankyrin稳定干涉后对小鼠乳腺癌转移具有明显的抑制作用.Gankyrin有可能成为新的肿瘤治疗靶点.
목적 건립은정간섭암기인Gankyrin적4T1-luc세포주,탐토Gankyrin대소서유선암전이적영향.방법 채용만병독감염화항성사선방법획득은정간섭Gankyrin적유선암세포주4T1-luc/shGankyrin,통과단백질인적화실시정량PCR방법분별재단백질화mRNA수평검측Gankyrin적간섭효과；선용BALB/c소서진행4T1세포원위충식,이용활체성상기술실시관측소서체내종류생장화종류전이정황,병대소서폐전이류진행병이학분석.결과 채용면역인적화실시정량PCR,검측은정고저Gankyrin 4T1세포재단백질수평화mRNA수평적표체,균명현저우대조세포,여대조세포상비,불동간섭서렬적4T1세포mRNA수평분별위대조세포적4.9％、25.1％、69.8％.이용활체성상진행세포수여세포발광강도적검측,선택세포수여발광강도기본일치적#2세포주진행소서원위충식,기산생적전이류진행실시관측,결과현시고저Gankyrin적4T1세포유도적폐전이류형광강도위3.02×106,이대조세포위10.9×106,동시병이학증실료대조세포산생적폐전이류면역조직화학Gankyrin염색양성,이고저Gankyrin적4T1세포적폐전이류면역조직화학Gankyrin염색음성.결론 채용만병독감염적방법가이유효건립은정고저Gankyrin표체적세포주；Gankyrin은정간섭후대소서유선암전이구유명현적억제작용.Gankyrin유가능성위신적종류치료파점.
Objective To establish Gankyrin knocking down 4T1-luc cell model and detect the effects of Gankyrin expression on breast cancer metastasis.Methods 4T1-luc cells carryring shGankyrin construct were established by lentivirus infection and antibiotic screening.Western blotting and real-time PCR were used to check the expression levels of Gankyrin.In vivo imaging system was used to monitor the effects of Gankyrin knocked down on cell growth and tumor metastasis after the in situ implantation of Gankyrin knocking down 4T1-luc cells in BALB/c mice.Results The cell expression decreased at the protein and mRNA levels.Gankyrin mRNA expression in different shGankyrin 4T1-luc cells was respectively 4.9％,25.1％ and 69.8％ versus the control cells.ShGankyrin#2 4T1-luc cells were chosen for in situ implantation into BAL/c mice because luminescent intensity was consistent with cell numbers.The photon flux of lung metastatic tumor induced by Gankyrin knocking down 4T1-luc cell was 3.02 × 106,while that of lung metastasis induced by control ceils was 10.9 × 106.The differences between two groups were significant.In pathology,Gankyrin was detected positive in lung metastasis tumors induced by control group.However,Gankyrin was negative in the Gankyrin knockdown group.Conclusions Lentivirus infection may be effectively used to establish Gankyrin knocking down 4T1-luc cell model.Because of its involvement in the in vivo pulmonary metastasis of breast cancers,Gankyrin should be a novel target for tumor therapy.